Drosophila SLC5A11 Mediates Hunger by Regulating K(+) Channel Activity

Abstract

Hunger is a powerful drive that stimulates food intake. Yet, the mechanism that determines how the energy deficits that result in hunger are represented in the brain and promote feeding is not well understood. We previously described SLC5A11-a sodium/solute co-transporter-like-(or cupcake) in Drosophila melanogaster, which is required for the fly to select a nutritive sugar over a sweeter nonnutritive sugar after periods of food deprivation. SLC5A11 acts on approximately 12 pairs of ellipsoid body (EB) R4 neurons to trigger the selection of nutritive sugars, but the underlying mechanism is not understood. Here, we report that the excitability of SLC5A11-expressing EB R4 neurons increases dramatically during starvation and that this increase is abolished in the SLC5A11 mutation. Artificial activation of SLC5A11-expresssing neurons is sufficient to promote feeding and hunger-driven behaviors; silencing these neurons has the opposite effect. Notably, SLC5A11 transcript levels in the brain increase significantly when flies are starved and decrease shortly after starved flies are refed. Furthermore, expression of SLC5A11 is sufficient for promoting hunger-driven behaviors and enhancing the excitability of SLC5A11-expressing neurons. SLC5A11 inhibits the function of the Drosophila KCNQ potassium channel in a heterologous expression system. Accordingly, a knockdown of dKCNQ expression in SLC5A11-expressing neurons produces hunger-driven behaviors even in fed flies, mimicking the overexpression of SLC5A11. We propose that starvation increases SLC5A11 expression, which enhances the excitability of SLC5A11-expressing neurons by suppressing dKCNQ channels, thereby conferring the hunger state.

Figure 1. Starvation enhances the excitability of SLC5A11 neurons, which requires SLC5A11 gene

(A) Representative traces of spontaneous activity recorded from the soma of SLC5A11 neurons in isolated brain preparations obtained from either 5-hr (fed, blue upper trace) or 22-hr (starved, red lower trace) food deprived adult fly. (Right) Enlarged firing patterns: regular spiking (top) or bursting (bottem). See also Figures S1 and S2. (B) Representative brain images of 5-hr (fed) or 22-hr (starved) deprived flies carrying P_SLC5A11-GAL4, UAS-mLexA-VP16-NFAT and LexAop-CD8-GFP-2A-CD8-GFP stained with the antibody to GFP in green and the neuropil marker nc82 in magenta, and the quantification of the intensity of GFP fluorescence in SLC5A11 neurons – the dotted boxes. Scale bar, 50 μm. See also Figure S3. (C) Representative traces of spontaneous activity and (D) quantification of firing frequency and bursting probability of SLC5A11 neurons in control and SLC5A11¹ mutant flies. See also Figure S2. (E) Behavior responses of 5-hr (fed) or 22-hr (starved) deprived control and SLC5A11¹ mutant flies in the two-choice assay. Flies were given a choice between 50mM D-glucose and 300mM L-glucose (n = 8-11) in this and subsequent behavior experiments unless indicated otherwise. f_event, event frequency; f_inst, instantaneous frequency. The numbers of bursting neurons out of the total number of recorded neurons are indicated above the bars. **P < 0.01, ***P < 0.001 vs. fed in the same genotype; error bars indicate SEM.

Figure 2. The activity of SLC5A11 neurons is necessary and sufficient for hunger-driven behaviors

(A) Representative traces of spontaneous activity and (B) quantification of firing frequency and bursting probability recorded from SLC5A11 neurons in the brains of fed flies carrying P_SLC5A11-GAL4 alone (control) and fed flies carrying P_SLC5A11-GAL4 and UAS-NaChBac (n = 12-13; ***P < 0.001). See also Figure S2. (C-E) Behavior responses of 5-hr (fed) or 22-hr (starved) food deprived flies carrying P_SLC5A11-GAL4 and either UAS-NaChBac (n = 8-12; ***P < 0.001 vs. fed in the same genotype) (C), UAS-dTrpA1 (n = 6-8; ***P < 0.001 vs. 22 °C in the same genotype) (D), or UAS-Shi^ts (n = 6-9; ***P < 0.001 vs. 22 °C in the same genotype) (E) in the two-choice assay. (F-H) Relative amounts of food consumed by 5-hr (fed) or 22-hr (starved) deprived flies bearing P_SLC5A11-GAL4, and either UAS-NaChBac (F,G) or UAS-Kir2.1 (H) (n = 9-17; ***P < 0.001 vs. control). (I,J) Olfactory responses of 5-hr (fed) or 22-hr (starved) deprived flies bearing P_SLC5A11-GAL4 or P_R38H02-GAL4 with either UAS-Kir2.1 (I) or UAS-NaChBac (J) to 1% ACV versus water in a T-maze preference assay (n = 7-15; ***P < 0.001 vs. fed in the same genotype). Flies harboring either P_SLC5A11-GAL4 or UAS-responder alone were used as controls. Error bars indicate SEM. See also Figure S4.

Fig. 3. Starvation increases the expression of SLC5A11 transcript

(A) Quantitative real-time PCR assay for SLC5A11 transcripts using the SYBR green method from the brains of flies that were food deprived for 5 hours (fed) and for 22 hours (starved), and had been deprived for 22 hours, but were refed for 24 hours (refed). The transcript levels in starved and refed flies are normalized to those in fed flies. GAPDH is used as a reference (n = 9, three independent biological replicates; ***P < 0.001 vs. fed). (B) SLC5A11 transcript levels of fed flies (fed), and 22-hr starved flies that were refed for 0, 10, 30, 60, 180, 300, 600 and 1,200 minutes (starved>>refed). The transcript levels of the starved>>refed flies are normalized to those in starved flies. The rate constant is predicted by a single exponential decay function. (C) Starvation-induced transcriptional activity of the SLC5A11 promoter in SLC5A11 neurons is visualized by monitoring de novo synthesis of green fluorescence in flies carrying P_SLC5A11-GAL4 and UAS-Kaede. After preexisting green fluorescence was photoconverted to red fluorescence, the flies were reared in either fed or starved conditions prior to fluorescence imaging. Scale bar, 20μm. (D) Relative amount of newly synthesized green KAEDE in SLC5A11 neurons in starved condition. The values were calculated by normalizing de novo KAEDE to preexisting red KAEDE (ΔF) followed by normalizing the measurements from starved condition to those from fed condition (%ΔF; n = 26-42; ***P < 0.001). Note that the control c42-Gal4 labeled neurons comprise a subset of R2 and R4 neurons. Error bars indicate SEM.

Fig. 4. SLC5A11 does not produce sugar-dependent co-transport currents

(A) Representative current traces in response to -100 mV test pulse from a holding potential of −50 mV in oocytes injected with H₂O, hSGLT1, or SLC5A11 cRNA. Only hSGLT1 expressing oocytes show glucose-dependent co-transport currents, which are blocked by the application of phlorizin, an antagonist of SGLT1. Corresponding time courses of peak current amplitude (right). (B) Quantification of current amplitudes recorded from oocytes injected with H₂O, hSGLT1, or SLC5A11 cRNA (n = 9-11; ***P < 0.001 vs. baseline). (C) Sugar- or monocarboxylate-dependent co-transport current and voltage relationship measured in oocytes expressing SLC5A11 or hSGLT1, exposed to different stimuli (n = 3-4). (D) Confocal images of oocytes injected with H₂O or EGFP-tagged SLC5A11 (upper). Current traces recorded from the corresponding oocytes (lower). Scale bar, 200μm. (E) Sequence alignment of the putative sugar-binding site of sodium/glucose co-transporter homologs in different species. See also Figure S5.

Fig. 5. The leak current is not required for hunger-driven food choice behavior and the excitability of SLC5A11 neurons

(A) Behavior responses of 5-hr (fed) or 22-hr (starved) deprived flies carrying P_SLC5A11-GAL4 and either UAS-SLC5A11 or UAS-SLC5A11^I94A,S380A in the two-choice assay (n = 6-10; ***P < 0.001 vs. fed in the same genotype). (B) Representative traces of spontaneous activity and (C) quantification of firing frequency and bursting probability in SLC5A11 neurons of fed flies carrying P_SLC5A11-GAL4 and either UAS-SLC5A11 or UAS-SLC5A11^I94A,S380A, and flies carrying P_SLC5A11-GAL4 only (control). (n = 12-13; *P < 0.05, ***P < 0.001 vs. control). Note that the bars from control group were reproduced for the purposed of comparison (see Figure 2B). f_event, event frequency; f_inst, instantaneous frequency. The numbers of bursting neurons out of the total number of recorded neurons are indicated above the bars. Error bars indicate SEM. See also Figure S2.

Fig. 6. Decreased dKCNQ channel activity mediated by SLC5A11 triggers hunger-driven food choice behavior

(A) Representative current traces and (B) current-voltage relationships in oocytes expressing dKCNQ only, dKCNQ + SLC5A11, and dKCNQ + hSGLT1. (C) Average values for the dKCNQ current amplitude measured at 40mV with oocytes injected with different amounts of the corresponding cRNA (n = 17-20; ***P < 0.001 vs. dKCNQ only). See also Figure S6. (D) Coimmnoprecipitation from HEK293 cells expressing both dKCNQ-Flag and SLC5A11-GFP by using anti-Flag (upper) or anti-GFP (lower) antibodies, followed by the Western analysis. The result is a representative of three independent experiments (n = 3). (E) Behavior responses of dKCNQ mutant and control WT flies (n = 6-7; ***P < 0.001 vs. fed in the same genotype) and (F) behavior responses of flies in which dKCNQ was knocked down in SLC5A11 neurons by the expression of dKCNQ RNAi using P_SLC5A11-GAL4 or P_R38H02-GAL4 (n = 7-9; ***P < 0.001 vs. fed in the same genotype) in the two-choice assay. These flies were starved for 5 hours (fed) or 22 hours (starved) and were given a choice between 50mM D-glucose and 220mM (E) or 270mM (F) L-glucose. Flies bearing either P_SLC5A11-GAL4, P_R38H02-GAL4, or UAS-responder alone were used as controls. Error bars indicate SEM.